BOLETÍN LATINOAMERICANO Y DEL CARIBE DE PLANTAS MEDICINALES Y AROMÁTICAS 17 (3): 249 - 258 (2018) © / ISSN 0717 7917 / www.blacpma.usach.cl

Artículo Original | Original Article montagnifolia: essential oil composition and biological properties

[Zaluzania montagnifolia: composición del aceite esencial y actividades biológicas]

Nemesio Villa-Ruano1, Yesenia Pacheco-Hernández2, Efraín-Rubio-Rosas3, José A. Zárate-Reyes3, Carlos J. Castro-Juarez1 & Sergio A. Ramírez-García1

1Universidad de la Sierra Sur, Ciudad Universitaria CP 70800, Miahuatlán de Porfirio Díaz Oaxaca, México 2Centro de Investigación en Biotecnología Aplicada, Tlaxcala, México 3Centro Universitario de Vinculación y Transferencia de Tecnología, Benemérita Universidad Autónoma de Puebla, Puebla, México Contactos | Contacts: Nemesio VILLA-RUANO - E-mail address: [email protected]

Abstract: The chemical composition of the seasonal essential oils (2015-2016) from the leaves and flowers of Zaluzania montagnifolia is presented. The chemical content of those oils showed quantitative and qualitative differences. Germacrene D (19.9-29.8%), camphor (12.4- 19.4%) and β-caryophyllene (13.7-18.5%) were the most abundant volatiles in the leaves. The essential oils from the flowers contained high amounts of camphor (32.7-37.2%) limonene (19.8-24.9%) and germacrene D (3.2-7.3%). All the seasonal essential oils showed a potent in -1 vitro inhibition against HMG-CoA reductase. The essential oils from flowers (IC50, 40.5-55.1 µg mL ) showed better inhibition properties -1 -1 -1 than those of leaves (IC50, 84.4-123.5 µg mL ). Camphor (IC50, 72.5 µg mL ) and borneol (IC50, 84.4 µg mL ) exerted a non-competitive inhibition on the enzyme. Additionally, the hydrodistillates exhibited antibacterial activity against the phytopathogenic Pseudomonas syringae pv. tabaci TBR2004 (MIC, 62.7-76.5 µg mL-1) P. syringae pv. tomato DC3000 (MIC, 45.4-50.4 µg mL-1) and P. syringae pv. phaseolicola NPS3121 (MIC, 26.7-31.9 µg mL-1). Germacrene D (MIC, 35.4-66.2 µg mL-1) and β-caryophyllene (MIC, 36.5-54.2 µg mL-1) were the strongest anti-Pseudomonas syringae agents.

Keywords: Zaluzania montagnifolia, seasonal essential oils, chemical profile, anti-HMG-CoA reductase, antibacterial.

Resumen: Se presenta la composición química de los aceites esenciales estacionales (2015-2016) provenientes de hojas y flores de Zaluzania montagnifolia. El contenido químico de los aceites esenciales mostró diferencias cualitativas y cuantitativas. El germacreno D (19.9-29.8%), alcanfor (12.4-19.4%) y β-cariofileno (13.7-18.5%) fueron los volátiles más abundantes en las hojas. Los aceites esenciales de las flores contuvieron altas concentraciones de alcanfor (32.7-37.2%), limoneno (19.8-24.9%) y germacreno D (3.2-7.3%). Todos los aceites esenciales estacionales mostraron una potente inhibición in vitro contra la HMG-CoA reductasa. Los aceites esenciales de las flores (IC50, -1 -1 40.5-55.1 µg mL ) mostraron mejores propiedades inhibitorias que aquellos de las hojas (IC50, 84.4-123.5 µg mL ). El alcanfor (IC50, 72.5 -1 -1 µg mL ) y el borneol (IC50, 84.4 µg mL ) ejercieron una inhibición no competitiva sobre la enzima. Adicionalmente, los hidrodestilados exhibieron una actividad antibacterial contra los fitopatógenos Pseudomonas syringae pv. tabaci TBR2004 (MIC, 62.7-76.5 µg mL-1) P. syringae pv. tomato DC3000 (MIC, 45.4-50.4 µg mL-1) y P. syringae pv. phaseolicola NPS3121 (MIC, 26.7-31.9 µg mL-1). El germacreno D (MIC, 35.4-66.2 µg mL-1) y β-cariofileno (MIC, 36.5-54.2 µg mL-1) fueron los agentes más fuertes contra los patovares de Pseudomonas syringae.

Palabras clave: Zaluzania montagnifolia, aceites esenciales estacionales, perfil químico, anti-HMG-CoA reductasa, antibacterial.

Recibido | Received: September 6, 2017 Aceptado | Accepted: November 1, 2017 Aceptado en versión corregida | Accepted in revised form: January 20, 2018 Publicado en línea | Published online: May 30, 2018 Este artículo puede ser citado como | This article must be cited as: N Villa-Ruano, Y Pacheco-Hernández, E-Rubio-Rosas, JA Zárate-Reyes, CJ Castro-Juarez, SA Ramírez- García1. 2018. Zaluzania montagnifolia: essential oil composition and biological properties. Bol Latinoam Caribe Med Aromat 17 (3): 249 – 258.

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INTRODUCTION halo blight diseases in Phaseolus vulgaris, The roots of Zaluzania montagnifolia () Lycopersicon esculentum, Nicotiana tabacum and are traditionally boiled to prepare an infusion for Solanum tuberosum (González et al., 2000) which are treating type 2 diabetes mellitus (DM2) in Oaxaca, considered as basic crops in rural communities in México (Turner, 2012). Previous reports described México. Thus, with traditional uses in this the aerial parts of the plant as a source of communities can be screened for their anti- sesquiterpene lactones (zaluzanins) and kaurene type phytobacterial properties in order to propose diterpenes with hypoglycemic, cytotoxic and alternatives for the biological control of uterotonic properties (Krishna-Kumari et al., 2003). phytophatogens. Due to the lack of scientific Kaurenoids and other small molecules dissolved in information regarding the volatile profile and the hexanic fraction of the plant also exhibited biological activities of the essential oil from Z. allelopathic, antioxidant and antibacterial activities montagnifolia, the principal objectives of this work (Villa-Ruano et al., 2013). Despite the pleasant aroma were to report the volatile variation in the essential of the leaves and flowers of Z. montagnifolia, there is oil from the plant and to determine the specific effect no available information on the chemical composition of selected volatiles on the normal activity of HMG- of its essential oil. Essential oils from CoA reductase. We additionally report on the medicinal/edible plants are a potential source of small antibacterial effect of the hydrodistillates on some molecules with pharmacological activity. The Pseudomonas syringae pathovars which are essential oils from Lippia turbinata, Origanum associated to severe infections of potato, tomato and vulgare and Salmea scandens showed interesting in bean. vitro activity against fat absorption (Quiroga et al., 2013; Villa-Ruano et al., 2015a). These facts suggest MATERIALS AND METHODS that natural products from medicinal plants could be Plant material used in the modulation of fat metabolism. Some Z. montagnifolia (SCH. BIP.) SCH. BIP. volatile organic compounds such as geraniol and (Asteraceae), was collected in Miahuatlán de Porfírio limonene were apparently able to decrease the Díaz, Oaxaca, México (16° 20.63’ N, 0.96° 34.85’ W, activity of 3-hydroxy-3-methylglutaryl CoA 1580 masl) during 2015-2016. Aerial parts of the reductase (HMG-CoA reductase), a key enzyme in assayed plants were previously certified in the cholesterol biosynthesis (Pattanayak et al., 2009). FCME-Herbarium at UNAM-México where a Thus, volatiles from medicinal plants can exert an reference voucher 130910 was deposited. improvement in the metabolism of cholesterol. Natural inhibitors of cholesterol biosynthesis are Chemicals found in traditional plants and their inclusion as an Analytical grade n-hexane was purchased from J.T alternative for the treatment of hypercholesterolemia Baker. Anhydrous sodium sulfate was from Bell is being envisioned (Lin et al., 2015). It is well ReactivesMR. The HMG-CoA reductase enzyme, known that high levels of LDL-cholesterol particles NADPH, 3-hydroxy-3-methylglutaryl CoA, are a risk factor for the development of DM2 and also resazurin, mixtures of n-alkanes and authentic that patients with DM2 maintain those levels when volatiles were all from Sigma-Aldrich Co. they are incorrectly treated (Mooradian, 2009). Therefore, the investigation of natural products from Isolation of the essential oils medicinal plants with potential use as inhibitors of Leaves of Z. montagnifolia were collected and dried HMG-CoA reductase should be investigated. in an oven at 30° C for 3 days in darkness. Seasonal On the other hand, alternative biological leaf samples were collected in spring (March 2015), activities of medicinal plant must be explored. summer (July 2015), fall (October 2015), winter Antibacterial activity of essential oils has been (January 2015) and spring 2016 (March 2016). extensively demonstrated in pathogens of human, Seasonal flower samples were only collected in the however, the effect of these preparations against spring periods, during March 2015 and March 2016. phytopathogenic bacteria has been poorly 300 g of dried leaves from five different plant investigated (Villa-Ruano et al., 2015a; 2015b). P. samples were extracted by hydrodistillation (five for syringae pathovars are one of the most prevalent each season, n=5) in a Clevenger type apparatus for 3 microorganisms causing the bacterial brown spot and h in order to obtain five different essential oil

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Villa-Ruano et al. Zaluzania montagnifolia and its biological activities samples. Posteriorly, the essential oils were recovered oils or pure volatiles (0.02, 0.08 and 0.14 mg mL-1) with n-hexane and immediately dried over anhydrous sodium sulfate. The density (mg mL-1) and yields of Antibacterial activity the essential oils (w/w) were obtained and stock The effect of the essential oils on the in vitro growth solutions (200 mg mL-1) were prepared to evaluate of Pseudomonas syringae pv. tabaci TBR2004, P. the hydrodistillates on HMG-CoA reductase and to syringae pv. tomato DC3000 and P. syringae pv. perform antibacterial tests (Villa-Ruano et al., phaseolicola NPS3121, was investigated by the broth 2015a). microdilution method using rezasurin as an indicator of cell viability (Sarker et al., 2007). The incubations GC-FID and GC-MS analyses were performed with different concentrations of the These procedures were done in a Hewlett Packard essential oil or authentic standards of germacrene D, 6890 II series with a HP-5 capillary column (30m X camphor, limonene and β-caryophyllene (10-500 µg 0.25mm I.D. covered with a 0.25 µm of 5:95 phenyl- mL-1) with 5X105 UFC in a 96-well plate in dimethylpolysiloxane as stationary phase). The GC- quintuplicate at a final volume of 0.3 mL. Dose- MS data were additionally corroborated in a Varian response curves (10-200 µg mL-1) were performed CP3800 gas chromatograph equipped with a capillary with Agrigen Plus® as a standard of reference. column Factor Four VF-5ms (30m X 0.25mm I.D, 25 Absorbance was measured at 545 nm. The MIC value µm of 5:95 phenyl-dimethylpolysiloxane as was considered as the initial concentration able to stationary phase) coupled to a Varian quadrupole avoid any absorbance change in the dose response 320MS model. The mobile phase was helium at 1 mL curves. The MIC values were presented as the min-1 flow rate. The GC-FID and GC-MS run average of the five essential oil samples obtained in conditions were adjusted in accordance with the each season studied. analytical procedures of a previous report (Villa- Ruano et al., 2015b). Metabolites were identified by RESULTS AND DISCUSSION comparison of their mass spectra (70 eV) with those The yields of the yellow crystalline essential oils of the NIST 2.0 Standard Reference Database, with from Z. montagnifolia was estimated in the range of the literature (Adams, 2007) and by co-injection with 3.5-4.2% (w/w) for the analyzed samples. Thirty-two available authentic standards. Kovats index (RI) was known compounds were identified in the essential calculated by simultaneous runs using a mixture of n- oils from the flowers and leaves of Zaluzania alkanes (C8-C20, C21-C40). The abundance of the montagnifolia (Table No 1). The abundance of metabolites was calculated on the basis of GC-FID sesquiterpenes in the leaves was higher (>50%) than peaks and the variation of the main volatiles was that of monoterpenes in the seasons studied (~40%). validated by ANOVA-Tukey Tests (p<0.01). In those hydrodistillates, germacrene D (19.9-29.8%) was the majoritarian sesquiterpene followed by Anti-HMG-CoA reductase activity camphor (12.4-19.4%) and β-caryophyllene (13.7- This activity was determined in each seasonal 18.5%). Significant fluctuations (p<0.01) in the essential oil (0.02-0.2 mg mL-1) by the relative abundance of germacrene D in leaves were spectrophotometric assays of NADPH oxidation observed along the analyzed seasons but, the described by Gholamhoseinian et al. (2010), with abundance of the other two majoritarian volatiles was slight modifications. The buffer described by those not significantly changed (Figure No 1). Contrary to authors was replaced by Tris-EDTA (0.1 M, pH 7.4) the essential oils from leaves, those from flowers and Pravastatin (Merck ®) was used as standard of showed a higher abundance of monoterpenes (~80%) inhibition. The IC50 was calculated from five than sesquiterpenes (~18%) (Table No 1). In this essential oils per season, then it was averaged and case, camphor (32.7-37.2%), limonene (19.8-24.9 %) presented as IC50 ± SD. The specific kind of and germacrene D (3.2-7.3%) were the most inhibition of the essential oils, camphor and borneol abundant volatiles. In the two flowering seasons was determined by double reciprocal Lineweaver- studied (spring 2015 and spring 2016), the three main Burk plot regressions performed with the GraphPad volatiles showed significant changes (p<0.01) (Figure Prims 5 software. The regressions were carried out No 2). The abundance of camphor is usually using distinct concentrations of HMG-CoA (0.1 - 2 conferred to leaves, shoots, stem barks, roots or mmol L-1) and three concentrations of the essential rhizomes of many plants but, its presence in flowers

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Villa-Ruano et al. Zaluzania montagnifolia and its biological activities is has been less reported (Mojab et al., 2007). Ruano et al., 2013). According to these results the Dramatic differences in the chemical composition essential oil of the plant could be considered as an between the analyzed essential oils and the hexanic alternative source for the obtainment of camphor and fractions from Z. montagnifolia were observed (Villa- germacrene D.

Figure No 1 Chemical variation of germacrene D, camphor and β-caryophyllene in the leaves of Zaluzania montagnifolia. Bars represent the standard deviation of five different seasonal samples. Diverse letters indicate statistically significant differences between means (p<0.01).

Figure No 2 Chemical variation of camphor, limonene and germacrene D in the flowers of Zaluzania montagnifolia. Bars represent the standard deviation of five different seasonal samples. Diverse letters indicate statistically significant differences between means (p<0.01).

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Table No 1 Chemical composition of the seasonal essential oils (2015-2016) from the leaves and flowers of Zaluzania montagnifolia. Leaves (%) Flowers (%) Compound cSP2015 cSU2015 cFA2015 cWI2015 cSP2016 RI1 b RI2 b cSP2015 cSP2016 anonane 0.3±0.08 0.9±0.12 - - 0.7±0.05 900 900 0.4±0.06 0.9±0.11 Tricyclene - - 0.3±0.04 1.5±0.18 0.9±0.12 923 921 0.3±0.05 - α-Thujene - - 0.7±0.02 1.3±0.05 0.6±0.08 931 933 0.3±0.07 - aα-Pinene 0.5±0.06 1.4±0.10 - 0.7±0.05 1.3±0.16 935 935 3.6±0.41 0.9±0.12 Camphene 0.7±0.02 2.8±0.34 - 1.5±0.17 2.6±0.05 952 954 3.8±0.46 0.6±0.04 Sabinene 0.8±0.13 - 0.6±0.02 0.9±0.12 - 971 973 - - aβ-Pinene 0.4±0.07 - - - 1.8±0.23 978 978 1.9±0.22 4.2±0.31 1-Octen-3-ol 0.7±0.09 2.6±0.17 - - - 980 981 2.8±0.31 3.1±0.39 β-Myrcene 0.6±0.04 - 0.3±0.04 - - 994 995 0.8±0.14 0.6±0.08 aLimonene 1.5±0.18 3.6±0.43 7.6±0.59 4.8±0.05 0.9±0.06 1030 1032 19.8±2.1 24.9±2.95 Ocimene 0.5±0.04 0.5±0.03 - - 1.1±0.14 1043 1044 0.6±0.08 - α-Terpinene - - 0.8±0.12 0.8±0.11 0.6±0.08 1063 1064 0.4±0.05 - Linalool oxide 0.3±0.08 2.8±0.06 - - 1.7±1.32 1074 1073 0.9±0.05 1.5±0.17 Terpinolene - - 0.4±0.06 0.4±0.05 0.8±0.09 1085 1086 0.6±0.08 - aLinalool 1.4±0.17 4.7±0.49 - 3.1±0.05 2.2±0.24 1098 1099 1.9±0.22 2.4±0.29 aCamphor 12.4±0.92 17.4±2.12 16.4±1.57 19.4±0.22 18.4±0.05 1138 1139 32.7±3.9 37.2±4.33 aBorneol 10.4±0.74 4.4±0.51 11.9±0.12 3.4±0.05 9.7±1.12 1166 1167 3.5±0.39 4.5±0.48 p-menth-1-en-8- ol 1.3±0.05 - - 0.3±0.02 - 1189 1187 0.8±0.09 1.3±0.15 Copaene 0.4±0.07 0.2±0.02 0.9±0.13 - - 1375 1376 0.8±0.11 - β-Cubebene 0.7±0.09 0.5±0.07 - - 0.3±0.05 1390 1391 - 0.7±0.04 β-Caryophyllene 16.5±1.8 18.5±1.52 14.5±1.23 15.5±0.14 13.7±1.56 1418 1417 2.4±0.25 3.4±0.38 Humulene 4.2±0.53 0.5±0.09 4.7±0.15 2.2±0.25 0.9±0.12 1449 1450 - 0.9±0.08 aGermacrene D 24.2±1.6 29.8±3.4 20.5±.1.8 26.3±2.9 19.9±0.05 1482 1483 7.3±0.85 3.2±0.37 α-Selinene 3.2±0.43 0.9±0.05 2.6±0.39 1.1±0.09 2.5±0.24 1489 1490 2.6±0.20 - Cadina-1(10),4- diene 0.5±0.07 - - 0.9±0.07 - 1524 1525 0.6±0.07 - Nerolidol 0.5±0.09 - - - - 1533 1533 0.2±0.05 0.8±0.09 Spathulenol 1.9±0.15 0.3±0.05 0.8±0.05 0.7±0.09 0.5±0.03 1571 1571 0.5±0.09 - Germacrene D-4- ol 6.2±0.90 1.2±0.05 3.2±0.22 9.4±1.1 4.1±0.42 1575 1576 2.1±0.25 3.1±0.39 tau.-Cadinol 1.2±0.09 0.9±0.03 2.6±0.34 - 1.7±0.15 1628 1629 0.4±0.01 - α-Bisabolol 1.4±0.08 0.4±0.06 0.4±0.06 - 0.7±0.02 1684 1684 0.6±0.09 0.3±0.04 Epimanool 2.6±0.07 0.6±0.08 3.1±0.43 3.4±0.21 3.1±0.37 1961 1962 2.5±0.29 3.5±0.41 Phytol 2.5±0.33 0.7±0.07 5.7±0.65 1.2±0.17 3.7±0.43 2112 2113 - Monoterpenes 31.5 40.2 39 38.1 42.6 74.7 81.2 Sesquiterpenes 60.9 53.2 50.2 56.1 44.3 17.5 12.4 Diterpenes 5.1 1.3 8.8 4.6 6.8 2.5 3.5 Hydrocarbons 0.3 0.9 0 0 0.7 0.4 0.9

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Total 97.8 95.6 98 98.8 94.4 95.1 98 a Compounds identified by co-injection with authentic standards. b RI1 and RI2, retention indices for HP-5ms and Factor Four VF-5ms columns, respectively. c SP spring; SU; summer; FA, fall; WI, winter. A dash (-) indicates no component identified

Table No 2 Anti-HMG-CoA activity of the seasonal essential oils from Zaluzania montagnifolia

aChemical agent bSP2015 bSU2015 bFA2015 bWI2015 bSP2016 Leaf essential oil 94.5±8.7 104.5±.9.2 115.4±6.5 84.4±7.8 123.5±9.7 Flower essential oil 55.1±4.5 - - - 40.5±3.9 Camphor 72.5±2.9

Borneol 84.4±2.6 Pravastatin 0.12±0.002 (IC50±SD) a -1 IC50 values are expressed in µg mL . bSP, spring; SU, summer; FA, Fall; WI, winter.

The seasonal essential oils from the leaves decreased well known, this compound is structurally related to the activity of the enzyme using approximately 0.1 camphor. Borneol was able to decrease Vmax to mg mL-1 (Table No 2). However, slight differences in 0.0085 mM min-1 (Figure No 3D). As non- the IC50 value were perceived in the essential oils of competitive inhibitors, camphor and borneol are able spring 2015 and winter 2015 (Table No 2). On the to cause a delay in the production of cholesterol other hand, the essential oils from the flowers (IC50, intermediates, thus the final production of the 40.5-55.1 µg mL-1) displayed a better inhibition rate triterpene will be decreased as the inhibitor interacts -1 than those of leaves (IC50, 84.4-123.5 µg mL ). with the enzyme. Remarkably, camphor and its Under standard conditions the Km of HMG-CoA for isomer borneol have been used as flavoring agents HMG-CoA reductase was 0.532 mM and the Vmax found in some plant foods such as basil, coriander, was 0.0116 mM min-1. The essential oils from both marjoram, rosemary and sage which are used since aerial organs produced a non-competitive effect on ancient times (Aguilar et al., 2008). Previous reports HMG-CoA reductase (Figure No 3 A-B). Vmax was revealed that other monoterpenes such as limonene reduced from 0.0116 to 0.0081 mM min-1, whereas exerted a potent inhibitory effect on the studied the Km parameter was not affected (0.531 mM). The enzyme (Pattanayak et al., 2009). However, this is the chemical profiles of the essential oils from the first work reporting the inhibitory effect of flowers, revealed camphor as the main constituent oxygenated camphor and borneol on the assayed (Table No 1). The enzymatic test with pure camphor enzyme. In spite of the potential toxicity of these suggested that this compound was strongly involved oxygenated monoterpenes (Aguilar et al., 2008), the in the in vitro inhibition of the enzyme (Table No 2). oral administration of small quantities of camphor -1, Camphor reduced Vmax from 0.0116 to 0.0065 mM and borneol in rats (<50 mg Kg bw) demonstrated -1 min (Figure No 3C), whereas the Km value was not that these compounds appeared in plasma after 2 significantly changed (0.530 mM). Interestingly, no hours (Sun et al., 2014). These studies additionally inhibitory effects on HMG-CoA reductase were suggested that camphor can be rapidly converted into observed for germacrene D or β-caryophyllene at the borneol, a less toxic compound. Thus, the presence of assayed concentrations (<0.2 mg mL-1). This fact these volatiles in rat plasma strongly suggest their strongly suggested that other compounds in the bioavailability to interact with different physiological essential oils were also participating to exert the targets. Recently, it was reported that the essential inhibitory activity. Borneol (3.4-10.4%) was tested oils from ginger and rosemary exerted a for the same activity observing its effect as an hypocholesterolemic effect in rats (Eissa et al., 2017). inhibitor of the studied enzyme (Table No 2). As is These oils contained camphor and borneol as

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Villa-Ruano et al. Zaluzania montagnifolia and its biological activities majoritarian volatiles. In accordance with our results camphor and borneol. Further in silico and previous literature, the strongest effect of the approximations should be required to predict the essential oils from the flowers of Z. montagnifolia interaction of those volatiles with HMG-CoA could be linked to the synergistic effect of limonene, reductase.

Figure No 3 Lineweaver-Burk plot regressions of the essential oils from Z. montagnifolia on the specific activity of HMG- CoA reductase. A, effect of the essential oil from leaves (Spring 2016). B, effect of the essential oil from the flowers (Spring 2016). C, effect of authentic camphor. D, effect of authentic borneol. The concentrations of HMG-CoA was in the range of 0.1-2 mmol L-1

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The antibacterial activity of the essential oils was 2015). Controversially, P. putida shows slight observed for the three assayed Pseudomonas syringae sensitivity to the action of camphor but not to that of pathovars. However, the seasonal essential oils were its isomer borneol (Prasad et al., 2011). These more effective on the growth inhibition of the evidences suggest that the assayed bacterial strains pathovar phaseolicola (Table No 3). The IC50 values could probably contain a similar biochemical were relatively higher for the pathovars tomato and biotransformation mechanisms to that of tabaci but, according to our results the less affected Pseudomonas putida (Prasad et al., 2011; bacterial strain was the pathovar tabaci (Table No 3). Loeschetcke & Thies., 2015). Germacrene D and β- In all the cases, the commercially available caryophyllene showed the best antibacterial activity antibiotics were more effective than the assayed in the three assayed pathovars (Table 4). The growth essential oils (Table 3). Nevertheless, the use of these inhibitory properties of β-caryophyllene has been oils could be envisioned as an alternative for the already demonstrated in Pseudomonas syringae pv. biological control of those phytopathogenic tomato (Huang et al., 2012). However, up to our microorganisms. The hydrodistillates from Z. knowledge this is the first report on the antibacterial montagnifolia showed a better in vitro inhibitory activity of germacrene D on P. syringae pathovars. effect than the essential oils from Clinopodium According to these evidences, our results suggest that macrostemum and Salmea scandens in the same the effect of the assayed essential oils is strongly bacterial species (Villa-Ruano et al., 2015a; 2015b). related to the abundance and synergistic activity of Interestingly, the assessment of the majoritarian several volatiles dissolved these hydrophobic terpenes revealed that camphor was not completely fractions. This finding could be considered as the involved in the anti-bacterial activity (Table No 4). initial approach for further in vivo assays with The high MIC value of camphor and limonene (̴ 0.3 diseased plants. Considering that these bacterial mg mL-1), suggested that these monoterpenes species are a threat for crops and for farmers’s produced a slight toxicity in the three pathovars of economy, the obtainment of new alternatives for their Pseudomonas syringae. This property has been biological control of P. syringae would be very observed in Pseudomonas putida which is able to valuable as a contribution to resolve this problem convert camphor and limonene into less aggressive (González et al., 2000). derivatives (Prasad et al., 2011; Loeschcke & Thies,

Table 3 Antibacterial activity of the seasonal essential oils from Zaluzania montagnifolia (MIC±SD).

***Bacteria bSP2015 bSU2015 bFA2015 bWI2015 bSP2016 Standard LEAF P. s. TBR 121.2±5.4 130.4±7.8 146.2±2.4 91.32±1.7 103.2±5.9 10.2±0.5€ P. s. DC 73.6±2.2 82.5±5.3 107.3±3.8 62.6±2.6 85.3±1.2 16.9±0.9€ P. s. NPS 54.7±1.3 63.7±2.2 95.3±2.3 52.4±1.9 54.5±1.4 15.8±0.7€ FLOWER P. s. TBR 76.5±0.4 62.7±0.3

P. s. DC 50.4±0.7 45.4±0.1

P. s. NPS 31.9±0.8 26.7±0.6 P.s. TBR, Pseudomonas syringae pv. tabaci TBR2004 P.s. DC, Pseudomonas syringae pv. tomato DC3000 P.s.NPS, P. syringae pv. phaseolicola NPS3121 € Ag, Agrigent Plus® used as antibiotic of reference *** MIC values are expressed in µg mL-1 bSP, spring; SU, summer; FA, Fall; WI, winter.

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Table No 4 Antibacterial activity of the majoritarian volatiles of the essential oil from Z. montagnifolia on Pseudomonas syringae pathovars (MIC±SD).

***Bacteria Camphor Germacrene D β-caryophyllene Limonene P. s. TBR 289.1±0.5 38.3±2.2 54.2±1.3 284.3±2.4 P. s. DC 321.6±2.2 35.4±4.9 36.5±3.8 299.4±1.5 P. s. NPS 324.6±2.6 66.2±3.6 39.2±3.9 276.7±1.8 P.s. TBR, Pseudomonas syringae pv. tabaci TBR2004 P.s. DC, Pseudomonas syringae pv. tomato DC3000 P.s.NPS, P. syringae pv. phaseolicola NPS3121 *** MIC values are expressed in µg mL-1

CONCLUSION 711. The chemical profile of the seasonal essential oils González AJ, Landeras E, Mendoza MC. 2000. from the aerial parts from Z. montagnifolia was Pathovars of Pseudomonas syringae causing described for the first time. Thirty two compounds bacterial brown spot and halo blight in were differentially detected in the studied years. Phaseolus vulgaris L. are distinguishable by Camphor and germacrene D were the most abundant ribotyping. Appl Environ Microbiol 66: 850 volatiles in all the seasons studied. The seasonal - 854. essential oils from the aerial parts showed anti-HMG- Huang M, Sanchez-Moreiras AM, Abel C, Sohrabi R, CoA reductase activity, camphor and borneol were Sungbeom L, Gershenzon J, Tholl D. 2012. evidently involved in such activity. The essential oils The major volatile organic compound emitted showed a relevant growth inhibitory activity on three from Arabidopsis thaliana flowers, the Pseudomonas syringe strains with special emphasis sesquiterpene (E)-β-caryophyllene, is a in the pathovar phaseolicola. Among the studied defense against a bacterial pathogen. New volatiles, germacrene D and β-caryophyllene were Phytol 193: 997 - 1008. the sesquiterpenes with the strongest antibacterial Krishna-Kumari GN, Masilamani S, Ganesh M, effect. Aravind S, Sridhar S. 2003. Zaluzanin D: a fungistatic sesquiterpene from Vernonia REFERENCES arborea. Fitoterapia 74: 479 - 482. Adams RP. 2007. Identification of essential oil Lin S-H, Huang K-J, Weng C-F, Shiuan D. 2015. components by gas chromatography/Mass Exploration of natural product ingredients as spectrometry. Allured, Carol Stream, IL, inhibitors of human HMG-CoA reductase USA. through structure-based virtual screening. Aguilar F, Autrup H, Barlow S, Castle L, Crebelli R, Drug Des Devel Ther 9: 3313 – 3324. Dekant W. 2008. Camphor in flavorings and Loeschcke A, Thies S. 2015. Pseudomonas putida—a other food ingredients with flavoring versatile host for the production of natural properties. Efsa J 729: 1 - 15. products. Appl Microbiol Biotechnol 99: Eissa FA, Choudhry H, Abdulaal WH, Baothman OA, 6197 – 6214. Zeyadi M, Moselhy SS, Zamzami MA. 2017. Mojab F, Tabatabai SA, Naghdi-Badic H, Nickavar Possible hypocholesterolemic effect of ginger B, Ghadyani F. 2007. Essential oil of the root and rosemary oils in rats. Afr J Tradit of Tanacetum parthenium (L.) Schulz. Bip. Complement Altern Med 14: 188 - 200. (Asteraceae) from Iran. Iran J Pharm Res 6: Gholamhoseinian A, Shahouzehi B, Sharif-Far F. 291 - 293. 2010. Inhibitory of some plant methanol Mooradian AD. 2009. Dyslipidemia in type 2 extracts on 3-hydroxy-3-methylglutaryl diabetes mellitus. Nat Clin Pract coenzyme A reductase. Int J Pharm 6: 705 - Endocrinol Metab 5: 150 - 159.

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